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Gains and strains of HCV diagnostics May 2001 Since 1990, when the first laboratory assay for detecting hepatitis C virus infection became available, remarkable progress has occurred in improving HCV diagnostics, defining HCV epidemiology, and increasing our understanding of the natural history of hepatitis C. The most dramatic progress has occurred in the area of public health, specifically in reducing the incidence of post-transfusion non-A, non-B hepatitis C from an estimated risk of one in 526 per blood unit in 1989,1 to one in 103,000 in 1996,2 and to virtually zero since routine HCV nucleic acid testing began in 1999. The Centers for Disease Control and Prevention estimated that approximately 300,000 persons living in the United States were infected with HCV via blood transfusion prior to 1990, when testing donated blood for HCV with immunoassays began.3 Now routine nucleic acid testing of all donated blood has essentially halted the spread of HCV by blood transfusion. Highlighted below are some of the impressive advances—and some of the unresolved issues—in the rapidly evolving field of HCV laboratory diagnostics. An innovative approach By 1975, serologic and epidemiological evidence had established the existence of a third hepatitis virus, provisionally termed the non-A, non-B agent. However, despite intensive efforts to isolate the putative NANB agent and develop a diagnostic assay, more than a decade passed before any of the candidate assays passed the litmus test of distinguishing between coded samples from uninfected controls and samples from pedigreed cases of NANB hepatitis. Investigators at Chiron Corp. and the CDC in 1989 announced the isolation of a cDNA clone derived from an NANB viral hepatitis genome.4 Bypassing the conventional first step of culturing the elusive virus to isolate a capture antigen for a diagnostic assay, these investigators adapted recombinant DNA technology to clone nucleic acid from an NANB-infected chimpanzee and expressed it in Escherichia coli and yeast. Subsequent studies using similar methods resulted in the development of three sequential generations of immunoassays for HCV antibodies, each offering incremental improvements in sensitivity and specificity. The innovative approach taken to identify a suitable capture antigen for the first HCV immunoassay pointed scientists in the direction of HCV molecular technologies, creating new opportunities for the development of HCV laboratory assays. Using molecular technologies, scientists at Chiron Corp. subsequently cloned the entire coding sequence of the HCV genome, leading to the production of an array of HCV-encoded recombinant and synthetic peptides for use in serologic assays for detecting HCV antibodies.5,6 Three generations of ELISAs for HCV antibodies The first-generation enzyme-linked immunosorbent assay for HCV antibody contained a single HCV recombinant antigen (c100-3) and was hurried to market in 1990 for testing donated blood to reduce the incidence of post-transfusion NANB hepatitis C. In 1992, a second-generation ELISA was developed that contained recombinant antigens from the virus’ nonstructural region (c100-3 and c33c) and an antigen (c22-3) from the core viral protein. This assay was more sensitive than the first-generation ELISA and became widely used as a clinical diagnostic and for epidemiological and other investigative purposes. More recently, a third-generation ELISA that has even greater sensitivity and that contains three recombinant antigens (c22-3, c200, and NS5) has been cleared by the FDA for screening donated blood (Ortho HCV Version 3.0 ELISA, Ortho-Clinical Diagnostics Inc., Raritan, NJ). Immunoblot assays Since the goal of HCV ELISA screening of donated blood was to eliminate potentially infective units from the blood supply, test algorithms and cutoff values for interpretations were established that prioritized sensitivity, recognizing that maximizing sensitivity would decrease specificity. Consequently, it became necessary to ensure that deferred donors received counseling about the meaning of their reactive HCV immunoassay results, which required that additional testing of their blood samples be conducted to identify false-positive results. For that purpose, a recombinant immunoblot assay, or strip immunoblot assay, was developed in which antibodies to specific HCV antigens could be detected and interpreted according to a standard algorithm. Three successive generations of recombinant immunoblot assays have evolved since 1990, each providing incrementally improved specificity for interpreting reactive HCV immunoassay results. The current third-generation RIBA uses three recombinant antigens (c33c, c100-3, and NS5) and one synthetic peptide from the core region (Chiron RIBA HCV 3.0 SIA, Chiron Corp., Emeryville, Calif.). Because the RIBA is based on the same recombinant antigens and synthetic peptides as the ELISA, it is licensed as "an additional, more specific test." "Confirmatory assay" does not appear on the FDA-cleared labeling. A "confirmatory" serological assay presumably will require capture antigens derived directly from HCV, or at least a source different from that of the immunoassay. Molecular assays The ability to detect HCV RNA with highly sensitive reverse transcription polymerase chain reaction has played an important role in confirming active HCV infection and in monitoring responses to treatment. While many laboratories have developed accurate and reliable in-house RT-PCR assays that can detect as few as 100 HCV RNA copies per milliliter of serum, others have been plagued by false-positive results due to contamination of test samples. Commercial test kits have eased clinical laboratories’ transition to nucleic acid testing. The kits include PCR-based assays (Amplicor HCV Monitor and COBAS Amplicor HCV Monitor, Roche Diagnostics Corp., Indianapolis), a transcription-mediated amplification assay (GenProbe Inc., San Diego), a branched DNA signal amplification assay (Quantiplex HCV RNA assay, Bayer, Emeryville, Calif.), and a nucleic acid-based signal amplification assay (Nuclisens, Organon Teknika, Durham, NC). The GenProbe assay is distributed as the Chiron Procleix HIV-1/HCV assay, which is designed to facilitate automated testing of donated blood in a combination format for detecting HCV and HIV-1 RNA. The Gen-Probe method amplifies RNA as the intermediate, whereas the Roche method uses RT-PCR to detect HCV or HIV RNA. Both methods have a sensitivity of approximately 100 genome copies per milliliter and are capable of detecting all six HCV genotypes. Quantitative tests for HCV RNA may be useful for monitoring the activity of HCV infections and how they respond to treatment. These tests include targeted amplification methods, such as qualitative PCR, that have been developed by research laboratories, as well as commercial kits, such as the Quantiplex HCV RNA or Amplicor HCV Monitor tests. Additional information about HCV immunodiagnostic and molecular assays for clinical laboratories can be found in the CAP practice parameter, "Testing for viral hepatitis."7 HCV genotypes and strains To develop even more sensitive assays to further decrease the risk of post-transfusion hepatitis continues to be a primary goal for HCV molecular diagnostics. But considerable progress also has been achieved in developing molecular technologies to analyze HCV genotypes and strains to support epidemiologic studies and tracing of HCV infections. Molecular and phylogenetic methods have been used to trace nosocomial spread of HCV during surgery, in hemodialysis units, and from HCV-contaminated intravenous immune globulin. Six distinct HCV genotypes can be identified by line probe-hybridization assay (Inno-Lipa, Immunogenetics, Zwijndrecht, Belgium) using type-specific probes. In one study, HCV strains belonging to genotypes 1 (subtypes 1a and 1b), 2, 3, 4, and 5 were characterized for 12 donor-recipient pairs implicated in cases of post-transfusion hepatitis C.8 Genetic distances and HCV mutation rates (0.6-2.1 x 10-3 per site per year) were determined. The sequences obtained from the 12 donor-recipient pairs clustered in 12 monophyletic nests. The results of this hybridization method were applied to exclude an HCV-infected donor as the source of HCV in an infected recipient who had been transfused with that donor’s blood. In another study, HCV genotyping with the Inno-Lipa hybridization method provisionally excluded an implicated donor typed as 1b as the source of HCV in a recipient typed as 1a.9 However, additional testing of sequence alignments in the nucleotide fragment of the HCV E1 region and construction of a phylogenetic tree demonstrated that the donor and recipient formed a monophyletic nest among HCV genotype 1a sequences, strongly linking the donor’s and recipient’s strains. This report highlights the limitations of interpreting hybridization studies to distinguish genotype 1 subtypes. Genotyping or phylogenetic analysis, or both, may prove to be useful for excluding an HCV-infected person as the source of another person’s HCV. It is highly improbable, however, that this technology will distinguish between individual strains, which is what would be required to establish a causal link between two HCV-infected persons. Nevertheless, molecular assays for HCV will support epidemiologic studies, forensic science, and even litigation, and will continue to grow and expand. Serological assays When the first-generation immunoassay for HCV antibody was licensed by the FDA and implemented in 1990, 0.5 to 0.6 percent of volunteer blood donors tested repeatedly reactive, and approximately 0.3 percent were considered to be truly positive—that is, RIBA-positive. When the second-generation HCV immunoassay was introduced in 1992, the RIBA-positive rate for volunteer donors was 0.16 percent.10 By 1996, the risk of transfusion-transmitted HCV had been reduced from the 1989 estimate of one in 526 to one in 103,000 per unit. Using data from the American Red Cross for 5 million repeat donors in 1998-99, the estimated risk of a transfusion-transmitted HCV infection was updated to one in 223,000.11 Donor notifications Testing donated blood for HCV antibodies by ELISA began in 1990 to protect recipients from acquiring transfusion-transmitted hepatitis C from HCV-infected donors. From the outset, in accordance with FDA requirements, all donated blood that tested repeatedly reactive for HCV antibody by ELISA was discarded and the donors were deferred indefinitely. Most, but not all, repeatedly reactive ELISA results were truly positive (RIBA-confirmed). Therefore, it was important to develop policies for informing donors whose HCV ELISA results were falsely positive that, although their blood was discarded and they were deferred indefinitely, they were unlikely to have hepatitis C. Beginning in 1991, the American Red Cross and some other blood collectors developed algorithms for HCV RIBA testing to issue specific and distinct notification messages to deferred donors with true-positive (RIBA-positive) HCV ELISA results but not to those donors with false-positive (RIBA-negative) HCV ELISA results. These early RIBA-based notification messages were, for the most part, readily understood by donors with true-positive ELISA results who were informed that they unknowingly had been infected with HCV. However, the message to donors with false-positive ELISA results—whose blood was discarded, who were indefinitely deferred, but who were told they were unlikely to be HCV-infected—was confusing and not readily clarified by an urgent visit to their health care provider. Even more confusing were the messages to those donors whose HCV RIBA results were indeterminate and who were informed that they may, or may not, be HCV-infected. During the past 10 years, I have counseled many deferred donors and witnessed the high price, in terms of peace-of-mind and physician and laboratory expenses, that some donors have paid as a result of blood safety initiatives for the benefit of patients. The availability of more sensitive and specific supplemental tests for HCV, including the RIBA 3.0 and HCV molecular diagnostic assays, has greatly improved the resources for diagnosing hepatitis C and distinguishing between true-positive and false-positive HCV ELISA results. It is hoped that a timely analysis of results from testing tens of thousands of blood donors’ samples every workday for alanine aminotransferase, antibodies to hepatitis B core antibody, anti-HCV, and HCV nucleic acid testing will establish the reliability of HCV nucleic acid testing (or HCV nucleic acid testing and anti-HCV) as an appropriate laboratory screen for detecting HCV-infected donors. Such an updated algorithm would not only eliminate the expense of continued testing for outdated surrogates for anti-HCV, but also could become the basis for retesting deferred donors and returning many of them to qualified donor status. For many former donors who were deferred because of false-positive surrogate or HCV ELISA results, questions about their health will nag them until their blood centers allow them to return as blood donors. Nucleic acid assays In March 1999, U.S. blood centers began implementing nucleic acid testing of donated blood to narrow the infectious seronegative window period by detecting early HCV and HIV-1 infection. GenProbe and Roche received FDA approvals for investigational new drug applications to develop unique, fast-track assays to do large-scale testing of donated blood. The American Red Cross selected the Gen-Probe technology in the multiplex HCV and HIV-1 format, initially referring all blood donors’ samples for testing to a laboratory in San Diego. Other blood collection facilities used various in-house PCR-based nucleic acid testing methods, or the Roche method, or referred blood donors’ samples to commercial laboratories. Following the format used when source plasma fractionators began nucleic acid testing in 1997, whole blood collectors also pooled donors’ samples before testing. In the United States, pool sizes varied from 16 to 128 samples, compared to pools of 96 to 1,200 samples for source plasma.12 Among the first 16.3 million donors tested by the American Red Cross, there were 62 HCV RNA-confirmed HCV-seronegative donations (1:263,000), a yield that was remarkably close to that predicted from the Red Cross’ data for 1998-99. These results reveal that nucleic acid testing brings blood transfusion safety even closer to the theoretical goal of "zero risk." At the same time, the results contribute to the ongoing controversy over the cost-effectiveness of increasingly smaller incremental benefits from new initiatives. Heading the list of nucleic acid testing-related issues is whether testing pools of donor samples, an expedient that was chosen for operational and cost considerations, should be phased out in lieu of resuming testing of individual blood samples. It has been shown that automated NAT for individual samples is technically feasible.13 Common sense, even without the support of a large-scale study, says that testing of individual samples has to be able to detect some low-titer samples containing HCV that would not be detected when diluted by testing pooled samples. Quantitative scientific data to support deliberations of this issue are being developed by the NAT Study Group under the auspices of the National Institutes of Health-sponsored Retrovirus Epidemiology Donor Study.14 Nationwide implementation of nucleic acid testing for donor blood using unlicensed assays has not been without controversy. On one hand, phase I implementation was conducted without requiring recipients’ specific informed consent to be transfused, if necessary, before nucleic acid testing results were available. That approach, approved by the NIH Office of Protection from Research Risks, presented hospitals with the prospect of an ethical dilemma, as well as potential litigation, if a blood component were to be transfused and, only a few hours later, reported by the blood center to be NAT-positive. Furthermore, most U.S. transfusion services were informed that neither the federal government nor the blood centers intended to fund this "research." Hospitals, such as Georgetown University Hospital, were surcharged $6 per unit of red cells by blood suppliers for the nucleic acid testing initiative, adding a significant, and largely nonreimbursable, expense. On the other hand, from an operational perspective, the roll-out of HCV nucleic acid testing was highly successful. Only one HCV NAT-positive component was transfused during the phase I implementation. Preliminary HCV-seronegative, NAT-positive detection rates reveal that approximately one in 263,000 units of infective whole blood were interdicted that otherwise would have been transfused. While it’s hard to argue with that level of success, at least one national leader, a former president of the American Association of Blood Banks, Laurence A. Sherman, MD, JD, believes that "legitimate questions exist about the merits of this costly endeavor relative to those of other public health projects...."15 HCV epidemiology The first specific data on the extent of HCV infection in the U.S. population emerged from early testing of blood donors using first-generation HCV immunoassays. While these data were important for blood collection operations, blood donor populations are not representative of the general population. Large-scale epidemiological studies, therefore, were necessary to estimate the true prevalence of HCV infection in the United States. Such population-based testing also was essential because most people with chronic HCV infection are asymptomatic; thus, estimates of HCV prevalence based on results of patient testing were also skewed. The most comprehensive U.S. study of HCV epidemiology was published by CDC investigators in 1999. Serologic and molecular methods were used to test blood samples for HCV. The samples were from 21,241 persons who had participated in the 1988-94 National Health and Nutrition Examination Survey.16 The results were staggering. An estimated 3.9 million people nationwide were estimated to have been infected with HCV (anti-HCV-positive). Of these, an estimated 2.7 million were chronically infected with HCV (HCV RNA-positive). Approximately 74 percent of these people were infected with HCV genotype 1 (1a or 1b), which is the most difficult HCV infection to treat. The most common risk factor in an earlier CDC study of acute HCV was a prior history of blood transfusion,17 but the health and nutrition survey, which captured lifelong acquisition of HCV, estimated that blood transfusion was a risk factor for only seven percent of the 3.9 million people who had been infected with HCV. The principal risk factors that were identified in the health and nutrition survey were surrogates for parenteral drug use—that is, cocaine or marijuana use—or surrogates for sexual transmission-that is, number of lifetime sexual partners, herpes simplex type 2 infection, or age at first intercourse. Natural history of HCV infection In 1989, when a limited number of HCV immunoassays became available for research studies, the first patients to be tested were already receiving medical care for symptomatic NANB hepatitis. Physicians from Japan, Spain, and Italy, among others, reported individual cases of progressive clinical and pathological deterioration from relatively mild acute hepatitis C to chronic hepatitis, cirrhosis, and, for some, hepatocellular carcinoma.18 This bias in case selection, together with early reports that anti-HCV was ineffective in clearing HCV because of rapidly evolving HCV quasi-species, established a dismal perspective at the outset for the natural history of HCV infection. Today, however, that outlook has brightened due to the results of long-term case-controlled studies of HCV infection, which support better outcomes. Hepatic failure secondary to chronic hepatitis C is the principal indication for liver transplant in the United States. Nevertheless, a recent review of the world literature on the natural history of HCV infection concluded that approximately 20 percent of those who are acutely infected will recover spontaneously, and approximately two-thirds of those in whom HCV infection persists (many receiving antiviral treatment) will likely have nonprogressive liver disease.18 The remaining one-third are at risk for progressive and potentially serious liver disease. HCV lookback Probably the most controversial policy related to HCV testing of donated blood has been "targeted lookback," which is the practice of notifying transfusion recipients that, although the blood they received met all safety requirements at the time of transfusion, it was collected from a person who subsequently was identified as having evidence of HCV infection. The rationale for such notifications is based in part on the assumption that the blood donor likely was infective for HCV at the time of donation and either was not tested for anti-HCV or was tested prior to 1992 by the relatively less sensitive first-generation HCV immunoassay. It also assumes that knowledge of HCV infection will cause these transfusion recipients to protect their health by avoiding risk behaviors, such as drinking alcoholic beverages, and help them to avoid spreading HCV. The effectiveness—or lack of effectiveness—of HCV targeted lookback is documented in a recent CDC report. After analyzing the results of a questionnaire mailed to all 198 U.S. blood collection facilities and 5,442 hospital transfusion services, CDC investigators concluded that this massive nationwide effort will have identified less than one percent of the 300,000 HCV-positive persons in the United States who may have acquired their infection through blood transfusion.3 Despite significant effort and expense, only about two percent of the 300,000 HCV-infected persons in the United States who might have benefited from this campaign were targeted for notification, and only about 0.5 percent were newly identified as HCV-infected as a result of the lookback. That’s a dismal outcome, by any standard, for an expensive, federally mandated public health initiative. As the director of a hospital transfusion service, I am particularly concerned that this was an "unfunded mandate"—that is, the costs for complying with the federal requirement by tracing and testing donors was borne by blood centers and hospital transfusion services. Persons being transfused in hospitals today are unfairly and unknowingly absorbing the costs of lookback policies that address the problems of patients who had transfusions more than a decade ago. Targeted HCV lookback appears to be another example of the public’s uniquely high standard for transfusion safety, which has evolved into policies that impose no practical limitation on effort or expense and may not offer any incremental improvement in the safety of the blood supply. References
1. Donahue JG, et al. The declining risk of post-transfusion hepatitis C virus infection. N Engl J Med. 1992;327:369-373. Dr. Sandler is professor of pathology and medicine, Georgetown University Medical Center, Washington, DC, and director of transfusion medicine, Georgetown University Hospital. He is a CA fellow. |
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